氧化型低密度脂蛋白诱导大鼠血管平滑肌细胞增殖及周期素D1的表达

背景:氧化型低密度脂蛋白可通过Ras/Raf/丝裂原活化蛋白激酶激酶,丝裂原活化蛋白激酶途径诱导血管平滑肌细胞增殖及细胞周期蛋白的表达.目的:观察胞外信号调节激酶是否参与了氧化型低密度脂蛋白诱导的血管平滑肌细胞周期推进以及周期素D1的表达.设计、时间及地点:对照观察细胞学实验,于2008-02/10在广州医学院完成.材料:人血浆低密度脂蛋白来自健康志愿者血清.SD大鼠由广州医学院实验动物中心提供.方法:采用超速离心法分离人低密度脂蛋白进行氧化修饰及鉴定.以酶消化法培养大鼠血管平滑肌细胞,采用免疫组织化学染色法鉴定.主要观察指标:在静止的血管平滑肌细胞中加入MEK1/2选择性抑制剂10 μmol/L PD98059和,或50 mg/L氧化型低密度脂蛋白,以CCK-8和细胞计数法测定细胞增殖,流式细胞术观察细胞周期,蛋白印迹检测周期素D1蛋白表达及胞外信号调节激酶的活性.结果:CCK-8和细胞计数法显示,氧化型低密度脂蛋白能明显诱导血管平滑肌细胞增殖,PD98059抑制氧化型低密度脂蛋白诱导的血管平滑肌细胞增殖,并且呈时间依赖性.流式细胞术分析表明,经氧化型低密度脂蛋白处理24 h后,血管平滑肌细胞从G0/G1期进入S期,S期细胞百分比明显增高(P<0.01),加入PD98059处理24 h后,血管平滑肌细胞由 G0/G1期向S期的推进受到抑制.蛋白印迹结果显示,氧化型低密度脂蛋白能明显诱导周期素D1蛋白表达及胞外信号调节激酶的磷酸化,PD98059抑制氧化型低密度脂蛋所诱导的周期素D1蛋白表达以及胞外信号调节激酶的磷酸化.结论:氧化型低密度脂蛋白能诱导血管平滑肌细胞增殖,促进细胞由G0/G1期向S期转换以及周期素D1表达,其作用部分是由胞外信号调节激酶所介导.     

痹肿消汤干预胶原诱导关节炎模型大鼠关节滑膜组织细胞外信号调节激酶1/2及细胞外信号调节激酶5的表达

背景：滑膜细胞信号转导异常是类风湿性关节炎的重要发病机制之一。中药复方痹肿消汤治疗类风湿性关节炎具有较好的临床疗效,但在细胞信号转导水平探讨痹肿消汤治疗类风湿性关节炎的作用机制是十分必要的。目的：观察中药复方痹肿消汤对胶原诱导关节炎模型大鼠滑膜细胞外信号调节激酶1/2,5表达的影响。方法：SD大鼠45只随机数字表法分为对照组、模型组、痹肿消汤组。胶原诱导关节炎模型大鼠,用痹肿消汤药液3.0～3.5mL灌胃,1次/d,连续14d。检测造模后第14,21,28天大鼠滑膜细胞外信号调节激酶1/2,5的磷酸化水平。结果与结论：造模后,大鼠滑膜细胞外信号调节激酶1/2,5的磷酸化表达随时间而增加（P〈0.05）。痹肿消汤治疗后,细胞外信号调节激酶1/2,5磷酸化表达水平则明显下降（P〈0.05）。提示痹肿消汤能抑制细胞外信号调节激酶1/2,5蛋白激酶的激活,从而抑制细胞外信号调节激酶1/2,5信号转导通路的活化,这可能是其阻抑类风湿性关节炎滑膜异常增殖、关节骨质侵蚀的重要机制之一。     

痹肿消汤干预胶原诱导关节炎模型大鼠关节滑膜组织细胞外信号调节激酶1/2及细胞外信号调节激酶5的表达

背景:滑膜细胞信号转导异常是类风湿性关节炎的重要发病机制之一.中药复方痹肿消汤治疗类风湿性关节炎具有较好的临床疗效,但在细胞信号转导水平探讨痹肿消汤治疗类风湿性关节炎的作用机制是十分必要的.目的:观察中药复方痹肿消汤对胶原诱导关节炎模型大鼠滑膜细胞外信号调节激酶1/2,5表达的影响.方法:SD大鼠45只随机数字表法分为对照组、模型组、痹肿消汤组.胶原诱导关节炎模型大鼠,用痹肿消汤药液3.0～ 3.5 mL灌胃,1次/d,连续14 d.检测造模后第14,21,28天大鼠滑膜细胞外信号调节激酶1/2,5的磷酸化水平.结果与结论:造模后,大鼠滑膜细胞外信号调节激酶1/2,5的磷酸化表达随时间而增加(P < 0.05).痹肿消汤治疗后,细胞外信号调节激酶1/2,5磷酸化表达水平则明显下降(P < 0.05).提示痹肿消汤能抑制细胞外信号调节激酶1/2,5蛋白激酶的激活,从而抑制细胞外信号调节激酶1/2,5信号转导通路的活化,这可能是其阻抑类风湿性关节炎滑膜异常增殖、关节骨质侵蚀的重要机制之一.     

脊髓胶质纤维酸性蛋白和磷酸化细胞外信号调节激酶在慢性神经痛发病中的作用

目的:探讨慢性坐骨神经挤压损伤大鼠脊髓组织中胶质纤维酸性蛋白和磷酸化细胞外信号调节激酶表达的改变。方法:实验于2003-06/09在军事医学科学院毒物药物研究所一室完成。雄性SD大鼠12只,随机分为2组,假手术组和慢性坐骨神经挤压损伤组,每组6只。按Bennett法,大鼠腹腔注射戊巴比妥钠40mg/kg后,假手术组暴露右侧坐骨神经不结扎,慢性坐骨神经挤压损伤组在该神经主干部位,用4-0铬制羊肠线松结扎4道。两组术后7和14d以von-Freyfilaments和冰水测定触痛及冷刺激反应,采用免疫组化法测定慢性坐骨神经挤压损伤大鼠脊髓组织中胶质纤维酸性蛋白和磷酸化细胞外信号调节激酶表达的改变。结果:12只大鼠全部进入结果分析。①术后7和14d,慢性坐骨神经挤压损伤组(术侧)触痛阈值分别下降80.3%和84.8%,冷刺激反应分别升高309.4%和336.2%,组间比较差异有显著性(P<0.01)。②术后14d,慢性坐骨神经挤压损伤大鼠双侧脊髓背角胶质细胞胶质纤维酸性蛋白和磷酸化细胞外信号调节激酶表达均有增加,手术侧分别增加246.4%和173.6%,与假手术组比较差异有显著性(P<0.01)。结论:慢性坐骨神经损伤后,中枢神经系统胶质细胞外信号调节激酶/丝裂原活化蛋白激酶通路激活,可能参与慢性神经痛的发病过程。     

脑源性神经营养因子对缺氧胚脑皮质神经元发挥保护作用的细胞内信号传导机制

目的:通过胚脑皮质神经元的体外培养,探讨遭受缺氧刺激时,脑源性神经营养因子(Brain derivedneurotrophic factor,BDNF)对神经元发挥保护作用时所启动的细胞内信号传导通路。方法:对胚鼠皮质神经元进行体外培养,提取不同缺氧时间点单纯缺氧的对照组及BDNF干预组的细胞蛋白,采用免疫印记(western blotting)方法检测两组神经元在细胞外信号调节激酶(extracellular signal-related kinase,ERK1/2)/磷酸化ERK1/2、蛋白激酶Akt/磷酸化Akt、P38丝裂原活化蛋白激酶(P38 Mitogen-activated protein kinase,P38MAPK)/磷酸化P38MAPK表达上的差别。结果:与单纯缺氧组比较,加入BDNF可引起磷酸化ERK1/2和磷酸化AKT表达的增强,而磷酸化的P38MAPK在两组均没有表达。结论:BDNF引起磷酸化ERK1/2及磷酸化AKT表达的上调可能为BDNF发挥对缺氧神经元保护作用的机理之一。     

去除蛋白激酶A磷酸化位点的线粒体融合素2基因与血管平滑肌细胞增殖(英文)

背景:线粒体融合素2基因作用于血管平滑肌细胞Ras蛋白,通过胞外信号调节蛋白激酶1/2通路抑制细胞增殖.线粒体融合素2基因氨基酸序列第442位丝氨酸为蛋白激酶A磷酸化位点,与其磷酸化状态密切相关,能参与其功能调控.目的:观察大鼠线粒体融合素2基因在去除蛋白激酶A磷酸化位点后对大鼠血管平滑肌细胞增殖的影响及其相关信号通路.方法:利用已构建的携带绿色荧光蛋白基因、线粒体融合素2基因和去除蛋白激酶A磷酸化位点的线粒体融合素2基因的3种重组腺病毒,感染大鼠主动脉血管平滑肌细胞,将其传代培养3～10代后以抽签法随机分为4组:①不加干预的对照组.②感染携带绿色荧光蛋白的对照组(Adv-GFP组).⑨感染携带线粒体融合素2基因的实验组(Adv-Mfn2组).④感染携带去除蛋白激酶A磷酸化位点的线粒体融合素2基因的实验组(Adv-Mfn2-PKA(△)组).激光共聚焦显微镜观察完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在细胞中的定位.Westernblot检测p-ERK1/2表达水平及完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在血管平滑肌细胞中的表达.MTT法绘制细胞生长曲线.结果与结论:完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在血管平滑肌细胞中均表达蛋白特异性条带.两种基因表达产物都主要分布于线粒体外膜.与对照组和Adv-GFP组相比,Adv-Mfn2组吸光度值在第3,4,5,6天都显著降低(P＜0.01＝,Adv-Mfn2-PKA(△)组吸光度值无明显变化.与对照组和Adv-GFP组相比,Adv-Mfn2组p-ERK1/2表达水平显著降低(P＜0.01＝,Adv-Mfn2-PKA(△)组无明显变化.提示去除蛋白激酶A磷酸化位点的线粒体融合素2基因定位于线粒体外膜,对血管平滑肌细胞的增殖无拮抗作用,对胞外信号调节蛋白激酶1/2通路无抑制作用.     

脊髓胶质纤维酸性蛋白和磷酸化细胞外信号调节激酶在慢性神经痛发病中的作用

目的：探讨慢性坐骨神经挤压损伤大鼠脊髓组织中胶质纤维酸性蛋白和磷酸化细胞外信号调节激酶表达的改变。方法：实验于2003-06／09在军事医学科学院毒物药物研究所一室完成。雄性SD大鼠12只，随机分为2组，假手术组和慢性坐骨神经挤压损伤组，每组6只。按Bennett法，大鼠腹腔注射戊巴比妥钠40mg／kg后，假手术组暴露右侧坐骨神经不结扎，慢性坐骨神经挤压损伤组在该神经主干部位，用4-0铬制羊肠线松结扎4道。两组术后7和14d以von-Frey filaments和冰水测定触痛及冷刺激反应，采用免疫组化法测定慢性坐骨神经挤压损伤大鼠脊髓组织中胶质纤维酸性蛋白和磷酸化细胞外信号调节激酶表达的改变。结果：12只大鼠全部进入结果分析。①术后7和14d．慢性坐骨神经挤压损伤组(术侧)触痛阈值分别下降80．3％和84．8％，冷刺激反应分别升高309．4％和336．20h，，组间比较差异有显著性(P(0．01)。②术后14d，慢性坐骨神经挤压损伤大鼠双侧脊髓背角胶质细胞胶质纤维酸性蛋白和磷酸化细胞外信号调节激酶表达均有增加，手术侧分别增加246．4％和173．6％，与假手术组比较差异有显著性(P&;lt;0．01)。结论：慢性坐骨神经损伤后，中枢神经系统胶质细胞外信号调节激酶／丝裂原活化蛋白激酶通路激活，可能参与慢性神经痛的发病过程。     

表皮生长因子受体活化与胰腺癌细胞解离关系的实验研究

目的 明确表皮生长因子受体(EGFR)活化参与胰腺癌细胞解离调节的分子机制.方法 通过免疫荧光法检测仓鼠高转移株(PC-1.0)和低转移株(PC-1)胰腺癌细胞中EGFR、活化(磷酸化)EGFR (p-EGFR)、活化(磷酸化)丝裂原活化蛋白激酶激酶2 (p-MEK1/2)及活化(磷酸化)细胞外信号调节激酶1/2 (p-ERK1/2)的表达变化及其与胰腺癌细胞解离状态变化的关系.结果 胰腺癌细胞解离因子(DF)明显诱导低转移株胰腺癌细胞(PC-1)中EGFR、p-EGFR、p-MEK1/2和p-ERK1/2的表达,同时诱导其细胞克隆解离.相反,AG1478(EGFR活化抑制剂)明显抑制高转移株胰腺癌细胞(PC-1.0)中EGFR、p-EGFR、p-MEK1/2和p-ERK1/2的表达,同时诱导PC-1.0细胞聚集成细胞克隆.结论 表皮生长因子受体活化后激活MEK/ERK信号通路,从而参与胰腺癌细胞解离的调节.     

Aβ1～42致老年性痴呆大鼠海马神经元细胞外信号调节激酶1,2的表达

目的:建立老年性痴呆大鼠模型,观察老年性痴呆大鼠海马神经元细胞外信号调节激酶1,2表达的变化,探讨其在老年性痴呆发病机制中的作用。方法:采用立体定向下双侧海马注射Aβ1~42,建立老年性痴呆动物模型,经Y型电迷宫试验测试其行为学,采用免疫组织化学、蛋白印迹等方法观察细胞外信号调节激酶1,2表达变化。结果:模型组大鼠学习记忆能力较对照组显著下降,免疫组化显示海马CA1区细胞外信号调节激酶1,2的免疫反应阳性神经元数目及细胞平均光密度值均显著减少,蛋白印迹显示模型组较对照组条带变细,灰度值变小,差异均有统计学意义(P<0.05)。结论:海马神经元细胞外信号调节激酶1,2表达的减少可能参与了老年性痴呆的发病机制。     

p38MAPK的转导机制及相关疾病

细胞不断接受外环境的变化 ,将化学信号经过细胞表面受体及细胞内信号转导途径传递到细胞质及细胞核内 ,通过合成相应的蛋白质对外环境的变化做出反应。丝裂原活化蛋白激酶 (MAPK)是细胞内重要的信号转导系统之一 ,其中 ,p38MAPK是MAPK家族中的重要成员 ,它是丝氨酸 /络氨酸激酶 ,其丝氨酸 /络氨酸残基可被磷酸化 ,主要参与介导应激反应 ,细胞外多种应激原如放射线、紫外光、热休克、高渗液 ,促炎因子 (如TNF -α、IL - 1)等都可引起细胞内蛋白激酶的连锁反应 ,从而影响细胞的转录、蛋白合成、细胞表面受体表达等生物效应。1　p38上…     

Cisplatin potentiates 1,25-dihydroxyvitamin D3-induced apoptosis in association with increased mitogen-activated protein kinase kinase kinase 1 (MEKK-1) expression

1,25-Dihydroxyvitamin D3 (1,25D3) exhibits potent antitumor activity in the murine squamous cell carcinoma (SCC) SCCVII/SF, and the combination of 1,25D3 with cisplatin (1,25D3/cisplatin) demonstrates even greater activity. Because these agents possess different mechanisms of cytotoxicity, studies were initiated to define the mechanism by which the combination displays enhanced activity. Median dose-effect analysis demonstrates that 1,25D3 and cisplatin act synergistically to inhibit SCC growth. When SCC cells were treated with 1,25D3 (10 nM) and/or cisplatin (0.5 microg/ml), greater caspase-3 activation was observed for the combination than for either agent alone. This suggests that the enhanced cytotoxicity is, at least in part, due to greater induction of apoptosis. No alterations in cellular platinum concentration or platinum-DNA adducts were observed for 1,25D3/cisplatin cotreatment compared with cisplatin treatment alone. Effects of the combination on cisplatin and 1,25D3 signaling pathways in adherent (nonapoptotic) and floating (apoptotic) cells were explored. Cisplatin induced p53 and its downstream targets, p21(Cip1) (p21) and Bax, in both cell populations. In contrast, 1,25D3 reduced p53, p21, and Bax to nearly undetectable levels in adherent cells. In the floating cells, 1,25D3 reduced levels of p53 and p21, but Bax expression was maintained at control levels. Expression of these proteins in cells treated with 1,25D3/cisplatin was similar to treatment with 1,25D3 alone. The two agents also had divergent effects on survival and stress signaling pathways. Phospho-extracellular signal-regulated kinase 1/2 and phospho-Jun levels increased after treatment with cisplatin but decreased after treatment with 1,25D3 and 1,25D3/cisplatin. Moreover, cisplatin decreased levels of mitogen-activated protein kinase kinase kinase (MEKK-1), whereas 1,25D3 up-regulated MEKK-1, and 1,25D3/cisplatin further up-regulated MEKK-1. We propose that the increased cytotoxicity for 1,25D3/cisplatin results from cisplatin enhancement of 1,25D3-induced apoptotic signaling through MEKK-1.     

A guanylate kinase/HSV-1 thymidine kinase fusion protein enhances prodrug-mediated cell killing

Herpes simplex virus thymidine kinase (HSVTK) with the guanosine analog ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system for gene therapy of cancer. Despite the broad application of the HSVTK/GCV approach, phosphorylation of GCV to its active state is inefficient such that high, myelosuppressive doses of GCV are needed to observe an antitumor effect. One strategy used to overcome the poor substrate specificity of HSVTK towards GCV (Km = 45 microM) has been to create novel forms of TK with altered substrate preferences. Such mutant TKs have shown benefit and are currently in clinical use. We describe here a second strategy to increase the amount of intracellular triphosphorylated GCV by involving the second enzyme in the GCV activation pathway, guanylate kinase (GMK). As a means to overcome the bottleneck of prodrug activation from the monophosphate to the diphosphate, we sought to combine both the critical HSVTK and GMK activities together. In this report we describe the construction of a fusion or chimeric protein of HSVTK and guanylate kinase, show data that demonstrate it confers a approximately 175-fold decrease in IC50 compared to HSVTK alone in response to ganciclovir treatment in stably transfected C6 glioma cells and finally, we present biochemical evidence of a kinetic basis for this improved cell killing.     

Identification of 4-anilino-3-quinolinecarbonitrile inhibitors of mitogen-activated protein/extracellular signal-regulated kinase 1 kinase

A high-throughput screen for Ras-mitogen-activated protein kinase (MAPK) signaling inhibitors identified two series (class 1 and 2) of substituted 4-anilino-3-quinolinecarbonitriles as potent (IC(50)s <10 nmol/L) mitogen-activated protein/extracellular signal-regulated kinase 1 (MEK1) kinase inhibitors. These compounds had cyanoquinoline cores, but differed in their respective aniline groups [1a, 1b: 4-phenoxyphenylaniline; 2a, 2b: 3-chloro-4-(1-methylimidazol-2-sulfanyl)aniline]. These compounds were competitive inhibitors of ATP binding by MEK1 kinase, and they had minimal or no effect on Raf, epidermal growth factor receptor (EGFR), Akt, cyclin-dependent kinase 4 (CDK4), or MK2 kinases at concentrations >100-fold higher than those that inhibited MEK1 kinase. Both class 1 and 2 compounds inhibited in vitro growth of human tumor cell lines. A class 2 compound (2b) was the most potent inhibitor of human tumor cell growth in vitro, and this effect was linked to distinct suppression of MAPK phosphorylation in cells. Compound 2b did not affect phosphorylation status of other kinases, such as EGFR, Akt, and stress-activated protein (SAP)/c-jun-NH kinase (Jnk); nor did it affect overall tyrosine phosphorylation level in cells. However, compound 2b did inhibit MEK1 phosphorylation in cells. Inhibition of MEK1 phosphorylation by 2b was not due to a major effect on Raf kinase activity, because enzyme assays showed minimal Raf kinase inhibition. We believe compound 2b inhibits kinase activity upstream of Raf, and thereby affects MEK1 phosphorylation in cells. Even with the dual effect of 2b on MEK and MAPK phosphorylation, this compound was well tolerated and significantly inhibited growth of the human colon tumor cell line LoVo (at 50 and 100 mg/kg BID, i.p.) in a nude mouse xenograft model.     

胸苷激酶1在白血病患者化疗过程中的临床意义探讨

目的利用免疫印迹增强化学发光法检测白血病患者化疗过程中血清胸苷激酶1(sTK1)水平,分析sTK1在评估白血病患者治疗效果以及对治疗方案的指导价值。方法分别对22例血液肿瘤治疗未缓解患者,20例治疗部分缓解患者,8例治疗完全缓解患者和29例健康人sTK1水平进行测定。结果未缓解组sTK1平均水平为(4.10±1.32)pmol/L,部分缓解组sTK1平均水平为(1.73±0.54)pmol/L,完全缓解组sTK1平均水平为(0.96±0.45)pmol/L,健康人对照组为(0.79±0.47)pmol/L。治疗未缓解患者组sTK1平均水平与完全缓解及部分缓解组有显著差异(P<0.01,P<0.01),且明显高于健康人组。完全缓解组肿瘤患者sTK1水平与健康对照组sTK1水平相差不大(P>0.05)。结论sTK1的含量可用于血液肿瘤化疗效果的评估,以及指导临床进行化疗方案的调整。     

Inflammatory bowel disease reveals the kinase activity of KSR1

Kinase suppressor of Ras-1 (KSR1) is a recently identified member of the EGFR-Ras-Raf-1-MAPK signaling pathway. A new study demonstrates that KSR1 protects intestinal epithelium from TNF-alpha-induced apoptosis, abrogating inflammatory bowel disease (IBD). Since its discovery, there has been disagreement as to whether KSR1 possesses intrinsic kinase activity. Using transgenic mouse models and genetically modified mouse colon epithelial cells, Polk and coworkers show that the kinase activity of KSR1 is off in normal colon epithelial cells, becoming activated only at the onset of IBD. They also provide strong evidence that KSR1 kinase activity is essential for anti-apoptotic protection of the intestinal epithelium. These new data in support of KSR1 as a kinase highlight an ongoing debate as to whether KSR1 does indeed serve as a specific kinase in transphosphorylating and transactivating c-Raf-1 toward MEK1.     

Clinical role of recurrently elevated macro creatine kinase type 1

The typical creatine kinase (CK) isoenzymes include CK-BB, CK-MB, and CK-MM. Macro CK type 1, one of the atypical CK enzymes, has been identified in human serum, but the clinical significance still remains uncertain. In our laboratory, 105 patients who expressed serum macro CK isoenzyme type 1 were identified from March 2004 to March 2007. We found that macro CK type 1 recurred after at least one month in 16 patients. Clinical diagnoses were myopathy in 14 patients, sepsis in one, and acute coronary syndrome in one. The averages of serum total CK and macro CK type 1 were 9,132 and 1,925 (U/L), respectively. The linear regression analysis between recurrent macro CK type 1 and total CK revealed a good correlation (y=3.5054x+2381.3, R(2)=0.7822, P<0.001). Three patients had critical illness, including one respiratory failure and two mortalities. Good linear correlation is documented between total CK and recurrent macro CK type 1. In conclusion, the macro CK type I isoenzyme recurred primarily in patients with myopathy.     

Sulindac independently modulates extracellular signal-regulated kinase 1/2 and cyclic GMP-dependent protein kinase signaling pathways

Colorectal cancer is the second leading cause of cancer mortality in the United States. Substantial human and animal data support the ability of nonsteroidal anti-inflammatory drugs to cause regression of existing colon tumors and prevent new tumor formation. The mechanism by which the nonsteroidal anti-inflammatory drug sulindac prevents tumor growth is poorly understood and seems complex as sulindac can modulate several growth-related signaling pathways. Sulindac metabolites simultaneously (a) increase cellular cyclic GMP and subsequently activate cyclic GMP-dependent protein kinase (PKG); (b) activate c-jun NH2-terminal kinase (JNK); (c) inhibit extracellular signal-regulated kinase 1/2 (ERK1/2); and (d) decrease beta-catenin protein expression at times and doses consistent with apoptosis. The purpose of this study was to determine if PKG, ERK1/2, JNK, and beta-catenin are independent targets for sulindac in vitro. Pharmacologic activation of PKG with YC-1 increases JNK phosphorylation and induces apoptosis in colon cancer cells without modulating ERK1/2 phosphorylation or beta-catenin protein expression. Inhibition of ERK1/2 with U0126 induces apoptosis but fails to activate JNK phosphorylation or down-regulate beta-catenin protein expression. Cotreatment with U0126 and YC-1 synergistically increases apoptosis in colorectal cancer cells and recapitulates the effects of sulindac treatment on ERK1/2, JNK, and beta-catenin. These results indicate that sulindac metabolites modulate ERK1/2 and PKG pathways independently in colon cancer cells and suggest that the full apoptotic effect of sulindac is mediated by more than one pathway. Using similar combinatorial approaches in vivo may provide more effective, less toxic chemopreventive and chemotherapeutic strategies. Such therapies could dramatically reduce the incidence and death rate from colorectal cancer.     

免疫印迹法测定乳腺肿瘤患者胸苷激酶

目的　探讨用免疫印迹发光法 ,检测乳腺肿瘤患者血清细胞质胸苷激酶 (TK1)的研究及临床应用。方法　利用抗胸苷激酶单克隆抗体 (anti TK1mAb)建立点免疫印迹发光法 ,分析乳腺肿瘤患者、非肿瘤患者和体检健康者血清TK1的水平。结果　术前的乳腺癌患者、乳腺良性肿瘤患者、非肿瘤患者和体检健康者血清TK1的水平分别为 (39.3± 33.9) ,(3.7± 3.9) ,(1.7± 0 .5 )和 (1.4±0 .5 )pmol/L。淋巴结转移的术前和术后乳腺癌患者、乳腺良性肿瘤患者血清TK1与体检健康者差异有显著意义 (P <0 .0 0 1,P <0 .0 0 1,P <0 .0 5 ) ;而淋巴结未转移的术后乳腺癌患者、非肿瘤患者血清TK1与体检健康者差异无显著意义 (P >0 .10 ,P >0 .0 5 )。通过对术后乳腺癌患者血清TK1检测发现 ,淋巴结未转移患者TK1水平明显低于淋巴结转移患者 (P <0 .0 0 1)。结论　该方法有较好的敏感性与特异性 ,用于血清TK1的检测可作为乳腺癌的辅助诊断、术后疗效观察和预后判断的方法之一